摘要
目的评价含RGD肽基因导入系统K16GRGDSPC(简记作K16-RGD)作为新型基因转染载体的可行性,并探讨转化生长因子β1(TGFβ1)基因转染骨髓基质干细胞(BMSCs)的表达能力。方法固相合成法合成K16-RGD。以6μgK16-RGD:2μgpcDNA3-TGFβ1(pTGFβ1)比例转染第三代兔BMSCs,转染72h后通过免疫组织化学染色和图像分析检测TGFβ1的瞬时表达水平;G418筛选2周形成阳性克隆,扩大培养后同法检测TGF-β1的稳定表达水平。通过酶联免疫吸附(ELISA)试剂盒检测TGF-β1的表达分析转染的量效关系和时效关系。结果瞬时转染组免疫组化部分细胞呈强阳性,稳定转染组几乎全部细胞呈强阳性并且持续表达4周以上。图像分析显示瞬时转染组和稳定转染组TGF-β1的表达均比对照组显著增强(P<0·01),且稳定表达组明显高于瞬时表达组(P<0·05)。ELISA法检测量效关系显示,转染体系中K16-RGD(μg)∶pTGFβ1(μg)为3∶1时TGFβ1有最高的表达效率;时效关系显示,稳定转染组TGFβ1的表达比瞬时表达组高(P<0·05)。结论含RGD肽基因导入系统K16-RGD作为基因转染载体是可行的,TGFβ1基因转染BMSCs可有效表达TGFβ1,为下一步利用K16-RGD作为载体构建载TGFβ1基因骨基质材料进行骨缺损修复研究奠定了基础。
Objective To evaluate the feasibility of K16GRGDSPC serving as a new vector for gene delivery and study the ability to express transforming growth factor βl gene of transduced rabbit bone marrow stromal cells in vitro. Methods The peptide was synthesized by solid- phase batch peptide synthesizer and characterized. The cultured 3rd generation rabbit bone marrow stromal cells （BMSCs） were transfected with the complexes of pcDNA3 - TGFβ1 （pTGFβ1） and peptide vector in the ratio of 1 to 3 in vitro. Immunohistochemistry staining and map analysis were performed to examine TGFβ1 gene expression. Enzyme- linked immuno- sorbent assay （ELISA） was performed to explore the dose- effect and time- effect relationship for higher gene transfection. Results Part cells of transient expression group and all cells of stable expression group showed strong positive activity for immunohitochemistry staining. TGFβ1 expression lasted more than 4 weeks. Map analysis indicated significant increase of TGFβ1 expression in BMSCs after gene transfer especially stable TGFβ1 gene transfer compared with control group. The gene transfection efficiency was highest when the proportion of pTGFβ1 to RGD peptide was 1 to 3 in mass. Conclusion RGD - containing peptide K16GRGDSPC can be used as a new kind of nonviral gene transfer vector. Transfected BMSCs can express active TGFβ1 effectively,which provides experimental foundations for further gene- modified bone tissue engineering study.
出处
《中国骨与关节损伤杂志》
2006年第9期717-719,共3页
Chinese Journal of Bone and Joint Injury
基金
国家自然科学基金项目(编号:30200063
30470483)
关键词
含RGD肽
TGFΒ1基因
转染
骨髓基质干细胞
RGD - containg peptide
TGFβ1 gene
Gene transfection
Bone marrow stromal cells
